This proposal is focused on the X-ray structure determination of a complex between the oxytocin receptor (OXTR), a member of the G Protein-Coupled Receptor (GPCR) family, and cholesterol, a ubiquitous lipid in mammalian cells known to modulate functions of a variety of membrane proteins. Significant efforts will be devoted to developments of technologies facilitating structure determination of membrane proteins through the use of the stabilizing effect of lipids. For a long time, the oxytocin system was linked with supporting two critical reproductive functions in mammals: uterine contraction during labor and milk ejection during lactation. Recently, a significant interest has emerged concerning the involvement of the OXTR in social and emotional behavior. The OXTR is activated by a cyclic nonapeptide hormone oxytocin. The OXTR has been demonstrated to have a remarkable sensitivity to the levels of cholesterol in the membrane. Depletion or addition of cholesterol reversibly transform the receptor between low (Kd>100 nM) and high (Kd~1 nM) affinity states. The main hypothesis of this proposal is that cholesterol modulates the activity of the OXTR by binding to a distinct allosteric site(s) thus inducing a change in the receptor conformation. The proposed structural and functional studies will take advantage of the Lipidic Cubic Phase (LCP) as a promising matrix for stabilizing and crystallizing difficult membrane proteins. The specific aims are: 1) Correlate the identity and amounts of natural lipids bound to GPCRs with successes of crystal nucleation and growth in LCP; 2) Establish the effect of cholesterol on modulating ligand binding affinities of the OXTR in LCP; 3) Obtain crystals of the OXTR in complex with cholesterol. For structural studies, we will follow the highly successful strategy of replacing the third intracellular loop of the receptor with T4 lysozyme combined with crystallization in LCP, which has recently yielded high resolution structures of human b2 adrenergic and adenosine A2A GPCRs. PUBLIC HEALTH RELEVANCE: This research project will develop new technologies that will accelerate the success rate of membrane protein structure determination and will attempt to solve a structure of the human oxytocin G protein-coupled receptor. Members of the G protein-coupled receptor family are involved in critical cellular and physiological processes and therefore are important drug targets. High resolution crystal structures give insights into mechanism of signal transduction and provide necessary templates for the rational design of a new generation of more efficient and safe drugs.